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The Modulatory Effect of Anthocyanins from Purple Sweet Potato on Human Intestinal Microbiota in Vitro Xin Zhang, Yang Yang, Zufang Wu, and Peifang Weng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00586 • Publication Date (Web): 15 Mar 2016 Downloaded from http://pubs.acs.org on March 15, 2016
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Journal of Agricultural and Food Chemistry
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The Modulatory Effect of Anthocyanins from Purple Sweet Potato on Human
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Intestinal Microbiota in Vitro
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Xin Zhang* , Yang Yang, Zufang Wu, and Peifang Weng
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Running header: Modulatory Effect of Anthocyanins on Intestinal Microbiota
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Department of Food Science and Engineering, School of Marine Sciences, Ningbo
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University, Ningbo 315211, P.R. China
∗
Corresponding author: Xin Zhang; E-mail address:
[email protected] ACS Paragon Plus Environment
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ABSTRACT: In order to investigate the modulatory effect of purple sweet potato
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anthocyanins (PSPAs) on human intestinal microbiota, PSPAs were prepared by
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column chromatography and their influence on intestinal microbiota was analyzed by
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monitoring the bacterial populations and analyzing short-chain fatty acid (SCFA)
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concentrations at different time points. The numbers (log10 cell/mL) of
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Bifidobacterium
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Clostridium histolyticum and total bacteria after 24 h of culture in anaerobic
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fermentation broth containing PSPAs were 8.44 ± 0.02, 8.30 ± 0.01, 7.80 ± 0.03, 7.60
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± 0.03 and 9.00 ± 0.02, respectively, compared with 8.21 ± 0.03, 8.12 ± 0.02, 7.95 ±
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0.02, 7.77 ± 0.02 and 9.01 ± 0.03, respectively, in the controls. The results showed
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that
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Lactobacillus/Enterococcus spp., inhibited the growth of Bacteroides-Prevotella and
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Clostridium histolyticum, and did not affect the total bacteria number. Total SCFA
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concentrations in the cultures with PSPAs were significantly higher than in the
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controls (P90%) were purchased from Shanghai Qibang Biological Co. (Shanghai,
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China). The AB-8 resin was purchased from Anhui Sanxing Chemical Co. (Bengbu,
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China). All solvents used for chromatography were of HPLC grade.
(M3G),
cyanidin-3,5-β-D-O-diglucoside
(C35G)
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To identify the peaks, solutions of C3G, M3G, C35G and C3R were prepared in a
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2% formic acid solution. Serial dilutions were generated in a 2% formic acid solution
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to produce following standard solutions for the standard curves: 100 µg/mL to 0.25
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µg/mL.
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PSP Samples. PSPs were obtained from a local market in Ningbo, China. Fresh
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samples were selected randomly, washed with running tap water, cut into pieces of
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approximately 0.5 cm, lyophilized, ground into powder, and sifted through a 100
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mesh sieve. The prepared samples were stored at -80 °C until use.
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Preparation of PSPAs. PSPAs were prepared from PSPs using a reported method,
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with some modifications.14 Briefly, 50.0 g of PSP powder were extracted with 1,600
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mL of acid-ethanol (HCl, 1.5 mol/L) at 80 °C for 60 min. Then, the extract was
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centrifuged at 4,500 g for 15 min, and the resulting insoluble residue was treated
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again. The supernatants were combined and concentrated. The resulting residue was
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dissolved with deionized water, filtered through a 0.45 µm cellulose filter and applied
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to a column (30 × 1.6 cm) of wet-packed AB-8 resin according to our previously
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established method. After reaching adsorptive saturation, the column was washed with
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2 bed volumes (BVs) of distilled water and then eluted with 4 BVs of an ethanol
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solution (60:40, v/v). The effluent of the ethanol solution was collected and
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concentrated, affording the PSPA extract.
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HPLC-ESI-MS/MS Analysis and Characterization of PSPAs. PSPA samples
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were analyzed on an Agilent Technologies 1200 series HPLC (Agilent Technologies,
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CA, USA) using a reported method, with a slight modification.15 Separation was
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achieved on a Zorbax Stablebond Analytical SB-C18 column (4.6 mm × 250 mm, 5
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µm, Agilent Technologies, USA). Elution was performed using the following gradient
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of mobile phase A (aqueous 2% formic acid solution) and mobile phase B (methanol):
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0-2 min, 10-20% B; 2-40 min, 20-55% B; 40-41 min, 55-80% B; and 41-45 min, 80%
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B. The quantification of different anthocyanins was based on the peak areas and
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calculated as equivalents of the standard compounds; all contents were expressed as
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milligrams per gram dry weight. The temperature of the column oven was set at 40 °C,
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the flow rate was set at 0.6 mL/min, and the injection volume was 5 µL.
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Low-resolution electrospray mass spectrometry was performed with a solariX ion
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trap mass spectrometer (Bruker Daltonics, MA, USA). The experimental conditions
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were as follows: ESI interface and nebulizer, 50 psi; dry gas, 15.0 psi; dry temperature,
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320 °C; MS/MS, scan from m/z 200 to 1500; ion trap, scan from m/z 200 to 1,500;
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source accumulation, 50 ms; ion accumulation time, 300 ms; flight time to acquisition
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cell, 1 ms; smart parameter setting (SPS) and compound stability, 50%; and trap drive
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level, 60%.
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In Vitro Fermentation of PSPAs. The effects of the PSPAs on human intestinal
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microbiota were investigated using our previously reported method, with slight
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modifications.16 Fecal samples were obtained from 8 healthy volunteers (4 females
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and 4 males, 25-30 years old) who did not have any history of gastrointestinal
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disorders and had not been treated with antibiotics for the last 6 months. Fecal slurries
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were prepared by mixing fresh fecal samples with autoclaved phosphate-buffered
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saline to yield 10% (w/v) suspensions. Anthocyanin samples were mixed with
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autoclaved nutrient basal growth medium at a final concentration of 1% (w/v). The
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basal nutrient medium (pH 7.0) contained peptone, 2.00 g/L; yeast extract, 2.00 g/L;
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NaCl, 0.10 g/L; K2HPO4, 0.04 g/L; KH2PO4, 0.04 g/L; MgSO4·7H2O, 0.01 g/L;
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CaCl2·6H2O, 0.01 g/L; NaHCO3, 2.00 g/L; hemin, 0.02 g/L; cysteine HCl, 0.50 g/L;
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bile salts, 0.50 g/L; resazurin, 1.00 mg/L; Tween 80, 2.00 mL/L; vitamin K1, 10.00 µL;
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and distilled water. The samples were then inoculated with 150 µL of fecal slurry
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(10%, w/v) with a manual homogenizer in an anaerobic atmosphere of 10% H2, 10%
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CO2 and 80% N2 at 37 °C (anaerobic incubator made in Shanghai, China). All
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experiments were repeated in triplicate.
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Enumeration of Bacteria by Fluorescent in Situ Hybridization. The
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enumeration of the bacteria was conducted using fluorescent in situ hybridization
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(FISH) as described in our previous report, with minor modifications.16 Briefly,
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culture samples (100 µL) were taken at 0, 6, 12 and 24 h, added to 300 µL of a filtered
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paraformaldehyde solution (4%, w/v), and fixed overnight at 4 °C to enumerate the
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bacteria. The fixed bacterial cells were washed twice with 400 µL of PBS,
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re-suspended in 600 µL of PBS/ethanol (1:1, v/v) and stored at -20 °C. Hybridization
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was performed using 16S rRNA-targeted oligonucleotide probes labeled with cyanine
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dye-3 (Cy3) fluorescent dye to enumerate specific bacterial groups, whereas DAPI
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was used to count the total number of bacteria. The probes used in our study were
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Bif164, Lab158, Bac303 and His150, and the probe names, target genera or species
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and probe sequences were listed in Table 1. An Axio Imager A1 epifluorescence
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microscope (Carl Zeiss, Göttingen, Germany) was used to count the bacterial cells. At
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least six random fields were counted on each slide, and the bacterial numbers were
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expressed as log10 cells per milliliter ± standard deviation (SD).
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Determination of SCFA and Lactic Acid Content. The analysis of SCFA and
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lactic acid content was performed using an Agilent 1100 series HPLC system with a
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Beckman Ultrasphere column (4.6 × 250 mm, 5 µm, Beckman Instruments Inc., CA,
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USA).17 Fermentation samples (50 µL) were collected after 24 h, centrifuged at 5,000
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g for 15 min, and then filtered through a 0.22 µm cellulose membrane. The mobile
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phase consisted of a 20 mM KH2PO4 solution (pH 2.5, A) and methanol (B) in the
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following gradient system: 0-16 min, 5% B; 16-30 min, 5 to 30% B; and 30-40 min,
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30% B. The flow rate was 0.8 mL/min, and the detector was set at 210 nm. The
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temperature of the column oven was set at 30 °C, and the injection volume was 20 µL.
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SCFA and lactic acid concentrations were calculated according to calibration curves
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of the respective authentic compounds, including formic, acetic, propionic, butyric
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and lactic acids. The analysis was conducted three times.
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Statistical Analysis. The obtained results were analyzed using SPSS version 16.0
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(SPSS Inc., Chicago, IL, USA). Any significant difference was determined by
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one-way analysis of variance (ANOVA) followed by Tukey test for multiple
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comparisons; a difference was considered statistically significant at P
